Unaltered expression of the major protease genes in a non‐virulent <i>recA</i>‐defective mutant of <i>Porphyromonas gingivalis</i> W83

Abaibou, Hafid; Ma, Qingjun; Olango, G. Jon; Potempa, Jan; Travis, James; Fletcher, Hansel M.

doi:10.1034/j.1399-302x.2000.150107.x

Cited by 23 publications

(44 citation statements)

References 35 publications

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“…While a reduction in Arg-X-and Lys-X-specific proteolytic activities was observed in P. gingivalis FLL92, transcription of the gingipain genes was unaltered in these mutants compared to that of the wild-type strain (Abaibou et al, 2001). A similar phenotype of the gingipain genes was also seen in P. gingivalis FLL32, a recA-and vimE-defective isogenic mutant that had reduced Arg-X-and Lys-X-specific proteolytic activities (Abaibou et al, 2000;Vanterpool et al, 2004). While there was a unique late onset of Arg-X-and Lys-X-specific proteolytic activity in P. gingivalis FLL92, there was little or no observed change of proteolytic activity in stationary-phase in P. gingivalis FLL93, a vimE-defective mutant (Vanterpool et al, 2004).…”

Section: Introductionsupporting

confidence: 49%

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83

et al. 2006

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The authors have shown previously that the vimA gene, which is part of the bcp-recA-vimA operon, plays an important role in protease activation in Porphyromonas gingivalis. The gingipain RgpB proenzyme is secreted in the vimA-defective mutant P. gingivalis FLL92. An important question that is raised is whether the vimA gene product could directly interact with the proteases for their activation or regulate a pathway responsible for protease activation. To further study the mechanism(s) of VimA-dependent protease activation, the vimA gene product was further characterized. A 39 kDa protein consistent with the size of the predicted VimA protein was purified. In protein–protein interaction studies, the VimA protein was shown to interact with gingipains RgpA, RgpB and Kgp. Immune sera from mice immunized with P. gingivalis immunoreacted with the purified VimA protein. Taken together, these data suggest an interaction of VimA with the gingipains and further confirm the role of this protein in their regulation or maturation.

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Section: Introductionsupporting

confidence: 49%

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83

et al. 2006

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“…A similar phenotype of the gingipain genes was also seen in P. gingivalis FLL32, a recA-defective isogenic mutant that had reduced Arg-X-and Lys-X-specific proteolytic activities (2). The 64-kDa RgpB partially processed proenzyme was secreted in both P. gingivalis FLL92 and FLL93 (19,23).…”

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confidence: 56%

Altered Gingipain Maturation in vimA- and vimE -Defective Isogenic Mutants of Porphyromonas gingivalis

et al. 2005

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We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA-and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membraneassociated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA-and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobic bacterium, is an important etiological agent of chronic periodontitis. Furthermore, accumulating data suggest that this organism is also associated with systemic diseases and complications including atherosclerosis, preterm births, and low-birth-weight babies (15). While several virulence factors are implicated in the pathogenicity of P. gingivalis, the proteolytic abilities of this organism have been considered to play the most significant role in its virulence (7,11,14,15,17). The major proteases, called gingipains, consist of an arginine-specific protease (Arg-gingipain [Rgp]) and lysine-specific protease (Lys-gingipain [Kgp]). The Rgp is encoded by two genes, rgpA and rgpB, while Kgp is encoded by a single gene, kgp. These proteases, which are both extracellular and cell associated, are required for growth of the organism and other housekeeping functions, which include processing enzymes for various cell surface proteins, including individual proteases (24). Moreover, the multiple pathogenic effects of the gingipains, such as degradation of complement and immunoglobulin, inactivation of cytokines and their receptors, aggregation of platelets, attenuation of neutrophil antibacterial activities, and increase in vascular permeability and blood clotting prevention, are well documented (14).We have reported previously that the vimA and vimE genes can modulate the phenotypic expression of the gin...

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“…Other proteins that may have a role in the coordination of export and attachment of the CTD family proteins are those of the bcp-recA-vimA, vimE, vimF, and porR loci (1,2,17,35,48,(56)(57)(58). Inactivation of recA affects the phenotypic expression and distribution of the gingipains, as does inactivation of vimA, vimE, vimF, and porR.…”

Section: Discussionmentioning

confidence: 99%

“…P. gingivalis strains were maintained by weekly passage on 10% (vol/vol) defibrinated, lysed horse blood in blood agar base no. 2 (HBA) (Oxoid, Basingstoke, England) incubated at 37°C in an anaerobic atmosphere of 5% H 2 , 80% N 2 , and 15% CO 2 . P. gingivalis strains were grown in batch culture in brain heart infusion broth (Oxoid) supplemented with 5 mg ml Ϫ1 cysteine, 5 g ml Ϫ1 hemin, and 5 g ml Ϫ1 menadione or grown in a BioFlo C30 bench-top chemostat (New Brunswick Scientific, Edison, NJ) at 37°C (pH 7.3 Ϯ 0.1) with a working volume of 365 ml.…”

Section: Methodsmentioning

confidence: 99%

The RgpB C-Terminal Domain Has a Role in Attachment of RgpB to the Outer Membrane and Belongs to a Novel C-Terminal-Domain Family Found in Porphyromonas gingivalis

et al. 2006

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Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the P. gingivalis outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain P. gingivalis mutant (YH522AB) and an Arg-and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.Porphyromonas gingivalis is a gram-negative anaerobe that is found predominantly in subgingival dental plaques associated with the destruction of the tooth's supporting tissues (14, 52; for reviews, see references 24, 26, and 32). P. gingivalis produces three major cysteine proteinases (gingipains), two of which, RgpA cat and RgpB (which are almost identical in sequence), are specific for Arg-Xaa cleavage (39, 49, 51) and one of which, Kgp cat , is specific for Lys-Xaa cleavage (34,38,50). The proteinases are encoded by rgpA, rgpB, and kgp and are produced as zymogens with a Sec-type leader peptide followed by a prodomain, the catalytic domain, and, in the cases of RgpA and Kgp, several sequence-related adhesin domains (50). The RgpA polyprotein is proteolytically processed to produce RgpA cat and several adhesins that are designated RgpA A1 , RgpA A2 , RgpA A3 , and RgpA A4 , while the Kgp polyprotein is processed to produce Kgp cat and the adhesins Kgp A1 , Kgp A2 , Kgp A3 , Kgp A4 , and Kgp A5 (32). The RgpA and Kgp proteinase and adhesin domains occur together as a surface-associated complex (32, 50), with relatively little of the complex found free in the extracellular milieu.RgpB is produced as a 736-amino-acid (aa) precursor with a Sec-type signal sequence that is predicted to be cleaved after Ala 24 and a prodomain cle...

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Unaltered expression of the major protease genes in a non‐virulent recA‐defective mutant of Porphyromonas gingivalis W83

Cited by 23 publications

References 35 publications

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83

VimA is part of the maturation pathway for the major gingipains of Porphyromonas gingivalis W83

Altered Gingipain Maturation in vimA- and vimE -Defective Isogenic Mutants of Porphyromonas gingivalis

The RgpB C-Terminal Domain Has a Role in Attachment of RgpB to the Outer Membrane and Belongs to a Novel C-Terminal-Domain Family Found in Porphyromonas gingivalis

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